Delivery systems for DNA vaccine are critically needed, in order to achieve successful clinical translation of gene-based immunotherapy against a wide range of diseases, including cancer and bacterial and viral infections. Biodegradable polymers present a promising avenue for DNA vaccine delivery;however, in vivo efficacy of the current delivery polymers is limited. Rational design of polymers for DNA vaccine delivery is difficult, largely due to a lack of understanding of detailed interactions between polymer carriers and the extremely complex immune system within the in vivo physiologic context. Our long-term goal is to develop biodegradable polymeric DNA vaccine carriers based on mechanistic understanding of the immune processes in vivo. We focus on a novel class of biodegradable poly (ortho esters) (POEs) that has shown preliminary efficacy in generating tumor-suppressing immunity in animals, and we propose the following two specific aims: (1) We will synthesize new pH-sensitive, biodegradable cationic block copolymers with defined structure and properties, and establish their efficacy for promoting DNA uptake, antigen production and presentation, and phenotypic maturation of antigen presenting cells. (2) We will investigate systematically the in vivo mechanisms of the block polymers carrying model DNA vaccines, quantify and track the time course of critical molecular and cellular events during immune activation, using novel multi-color flow cytometry and immunostaining assays that enable us to track, simultaneously, multiple cellular markers in response to both CD8 and CD4 epitopes. Furthermore, we will examine the activation of endogenous na[unreadable]ve T cells in unperturbed in vivo context that is clinically relevant. Public Health Relevance: Combining a versatile and tunable biodegradable polymer system with advanced immunological tools and focusing on in vivo investigation, we hope to address important questions regarding how the timing, location, magnitude, and cells of immune processes may be optimally manipulated through better engineering of polymer delivery vehicles that will eventually lead to improved in vivo efficacy of DNA-based immunotherapy.